A novel role of the aryl hydrocarbon receptor (AhR) in centrosome amplification - implications for chemoprevention

Abstract

Background: Centrosome aberrations can cause genomic instability and correlate with malignant progression in common human malignancies such as breast and prostate cancer. Deregulation of cyclin/cyclin-dependent kinase 2 (CDK2) activity has previously been shown to be critically involved in centrosome overduplication. We therefore test here whether small molecule CDK inhibitors derived from the bis-indole indirubin can be used to suppress centrosome aberrations as a novel approach to chemoprevention of malignant progression.

Results: As expected, we found that the CDK inhibitor indirubin-3'-oxime (IO) suppresses centrosome amplification in breast cancer cells. However, we made the unexpected discovery that indirubin-derived compounds that have been chemically modified to be inactive as kinase inhibitors such as 1-methyl-indirubin-3'-oxime (MeIO) still significantly reduced centrosome amplification. All indirubins used in the present study are potent agonists of the aryl hydrocarbon receptor (AhR), which is known for its important role in the cellular metabolism of xenobiotics. To corroborate our results, we first show that the coincidence of nuclear AhR overexpression, reflecting a constitutive activation, and numerical centrosome aberrations correlates significantly with malignancy in mammary tissue specimens. Remarkably, a considerable proportion (72.7%) of benign mammary tissue samples scored also positive for nuclear AhR overexpression. We furthermore provide evidence that continued expression of endogenous AhR is critical to promote centriole overduplication induced by cyclin E and that AhR and cyclin E may function in the same pathway. Overexpression of the AhR in the absence of exogenous ligands was found to rapidly disrupt centriole duplication control. Nonetheless, the AhR agonists IO and MeIO were still found to significantly reduce centriole overduplication stimulated by ectopic AhR expression.

Conclusions: Our results indicate that continued expression of endogenous AhR promotes centrosome amplification in breast cancer cells in a pathway that involves cyclin E. AhR agonists such as indirubins inhibit centrosome amplification even when stimulated by ectopic expression of the AhR suggesting that these compounds are potentially useful for the chemoprevention of centrosome-mediated cell division errors and malignant progression in neoplasms in which the AhR is overexpressed. Future studies are warranted to determine whether individuals in which nuclear AhR overexpression is detected in benign mammary tissue are at a higher risk for developing pre-cancerous or cancerous breast lesions.

Figure 1

Indirubins inhibit centriole overduplication in breast cancer cell lines. (A) Immunofluorescence microscopic analysis of HCC1806 breast cancer cells for centrin to visualize normal centrioles (top panels) in contrast to centriole overduplication (bottom panels). Nuclei stained with DAPI. Scale bar indicates 10 μm. (B, C) Quantification of the proportion of HCC1806 or MCF-7 cells manipulated to stably express empty vector (MCF-7/neo) or cyclin E (MCF-7/cyclin E) with aberrant centriole numbers (>4 per cell) after treatment with 0.1% DMSO as solvent control or 1 μM indirubin-3'-oxime (IO), 1 μM 1-methyl-indirubin-3'-oxime (MeIO), 1 μM 6-bromo-indirubin-3'-oxime (BIO), 1 μM 1-methyl-6-bromo-indirubin-3'-oxime (MeBIO) or 1 μM 6-bromo-indirubin-3'-acetoxime (BIA) for 24 h. Each bar represents mean and standard error of at least three independent experiments with a minimum of 50 cells counted per experiment. Only mononucleated cells were assessed for centriole aberrations to exclude polyploid or otherwise altered cells.

Figure 2

The AhR is overexpressed in non-malignant and malignant breast tissue. Examples of an immunofluorescence microscopic analysis of non-malignant and malignant breast tissue specimens for nuclear overexpression of the AhR. Note the nuclear staining in the hyperplastic tissue specimen (second row from the top) as well as in the dysplasia and carcinoma. Nuclei stained with DAPI. Scale bar indicates 100 μm.

Figure 3

Centrosome aberrations in breast tissue. Examples of an immunofluorescence microscopic analysis of non-malignant and malignant breast tissue specimens for the centrosome marker γ-tubulin. Note the presence of extra centrosomes in the dysplastic and cancerous tissue specimens. Arrows in inset point to centrosomes. Nuclei stained with DAPI. Scale bar indicates 50 μm.

Figure 4

Knock-down of the AhR reduces centriole aberrations in breast cancer cell lines. (A, B) Immunoblot analysis of HCC1806 cells for AhR expression after transient transfection of cells with siRNA targeting the AhR or control siRNA duplexes. Immunoblot for Actin is shown to demonstrate protein loading (A). Quantification of the proportion of HCC1806 cells with aberrant centriole numbers following transfection with siRNA targeting the AhR or control siRNA. Each bar represents mean and standard error of at least three independent experiments with a minimum of 50 cells counted per experiment (B). (C, D) Immunoblot analysis of MCF-7/neo and MCF-7/cyclin E cells for AhR expression after transient transfection of cells with siRNA targeting the AhR or control siRNA duplexes. Immunoblot for actin is shown to demonstrate protein loading (C). Quantification of the proportion of cells with aberrant centriole numbers following transfection with siRNA targeting the AhR or control siRNA. Each bar represents mean and standard error of at least three independent experiments with a minimum of 50 cells counted per experiment (D). (E) Immunoblot analysis of MCF-7/neo and MCF-7/cyclin E cells following transient transfection (72 h) with either control siRNA (siControl) or siRNA targeting the AhR (siAhR). Immunoblots for AhR, cyclin E, cyclin A, p27^Kip1 and actin are shown. (F) Quantification of the proportion of MCF-7/neo cells (open bars) and MCF-7/cyclin E cells (black bars) with n > 4 centrioles following transient transfection with either control siRNA (siControl) or siRNA duplexes targeting CDK2 (siCDK2), cyclin E (siCyclin E), AhR (siAhR), CDK2 and AhR (siCDK2/siAhR) or cyclin E and AhR (siCyclin E/siAhR). Each bar represents mean and stardard error of two independent experiments each with triple quantification of at least 50 cells.

Figure 5

Overexpression of the AhR stimulates centriole overduplication that can be reverted by indirubins. (A) Fluorescence microscopic analysis of MCF-7 cells stably expressing centrin-GFP to visualize individual centrioles after transient transfection of cells with AhR or empty vector (control). Note the presence of two daughter centrioles at single maternal centrioles indicating aberrant daughter centriole synthesis (right panel). Arrows in right inset point to extra centrioles in an AhR-transfected cell. (B) Quantification of the proportion of MCF-7/centrin-GFP cells with aberrant centriole numbers following transfection with AhR or empty vector (control). Each bar represents mean and standard error of at least three independent experiments with a minimum of 50 cells counted per experiment. (C) Quantification of the proportion of MCF-7/centrin-GFP cells with n > 4 centrioles following transient transfection with either empty vector control (open bars) or AhR (black bars) and treatment with 0.1% DMSO, 1 μM IO or 1 μM MeIO starting 24 h after transfection of cells for an additional 24 h. Each bar represents mean and standard error of at least triple quantification of at least 50 cells.